Human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) are a promising source
for cell transplantation into the damaged heart, which has limited regenerative ability.
Many methods have been developed to obtain large amounts of functional CMs from hPSCs
for therapeutic applications. However, during the differentiation process, a mixed
population of various cardiac cells, including ventricular, atrial, and pacemaker
cells, is generated, which hampers the proper functional analysis and evaluation of
cell properties. Here, we established NKX2-5eGFP/w and MLC2vmCherry/w hPSC double
knock-ins that allow for labeling, tracing, purification, and analysis of the development
of ventricular cells from early to late stages. As with the endogenous transcriptional
activities of these genes, MLC2v-mCherry expression following NKX2-5-eGFP expression
was observed under previously established culture conditions, which mimic the in vivo
cardiac developmental process. Patch-clamp and microelectrode array electrophysiological
analyses showed that the NKX2-5 and MLC2v double-positive cells possess ventricular-like
properties. The results demonstrate that the NKX2-5eGFP/w and MLC2vmCherry/w hPSCs
provide a powerful model system to capture region-specific cardiac differentiation
from early to late stages. Our study would facilitate subtype-specific cardiac development
and functional analysis using the hPSC-derived sources.